[unreadable] [unreadable] Myotonic dystrophy is the second most common cause of muscular dystrophy and the most common cause of the adult-onset type. The major form, DM type 1 (DM1), is caused by a CTG expansion in the 3' untranslated region (UTR) of the DMPK gene, which is transcribed and forms nuclear RNA foci. Expression of expanded RNA (DMPK-CUG) in mice and in cell culture reproduces cellular and molecular aspects of DM1. The mutant RNA reverses a developmentally regulated alternative splicing transition, directly causing the myotonia and insulin resistance that patients experience. However, the muscle wasting, which is responsible for the most common cause of DM1 mortality and morbidity, has yet to be explained. Cultured myogenic satellite cells from patients exhibit a significant differentiation defect likely to be directly relevant to this symptom. This project will: (i) establish inducible mouse C2C12 myoblast cell lines that express DMPKCUG RNA to investigate the mechanism by which mutant RNA inhibits differentiation, and (ii) develop a novel approach to reverse these effects using antisense oligonucleotides (ASOs) and endogenous RNase H activity. In Specific Aim 1, C2C12 cell lines inducibly expressing RNA containing 960 CUG repeats in the context of the DMPK 3' UTR (DMPK-CUG960) will be generated using existing tetracycline transactivator lines and tet-responsive construct. Clonal lines with low basal expression, high induction, and robust differentiation will be identified; the ability of DMPK-CUG960 RNA to inhibit differentiation will be confirmed and the cultures will be analyzed for cellular and molecular features of DM1. In Specific Aim 2, these lines will be used to investigate the mechanism by which DMPK-CUG960 inhibits skeletal muscle differentiation. DMPK-CUG960 RNA will be induced before or after differentiation to determine the effects on myoblasts and post-differentiation myotubes; analysis of myogenic and cell cycle factors will be used to determine the point at which myoblast differentiation is blocked. In Specific Aim 3, chimeric locked nucleic acid ASOs and ubiquitous RNase H activity will be used to degrade DMPK-CUG960 RNA in these lines to restore muscle differentiation. Optimized ASOs will be assayed in DM1 cell cultures and in a mouse model expressing DMPK-CUG960 RNA developed in the sponsor's lab for their ability to reverse the striking muscle wasting. The goal of this project is to elucidate the mechanism of muscle wasting in Myotonic Dystrophy, the most common cause of adult-onset muscular dystrophy, and to reverse this symptom in cell culture and a mouse model. These investigations may also help understand other disorders such as Fragile X Syndrome and the related FXTAS Syndrome, Huntington's chorea, Friedreich's Ataxia, and Spinal Cerebellar Ataxia. [unreadable] [unreadable] [unreadable]